DEVELOPMENT AND CHARACTERIZATION OF DOXORUBICIN AND siRNA ENCAPSULATED CHITOSAN NANOPARTICLES

Objective: Chitosan nanoparticles (ChNP’s) have been widely studied for drug and gene delivery. In this study, we prepared ChNP’s for co-delivery of doxorubicin (DOX) and siRNA for cancer treatment. Methods: The ionic gelation method was used to develop ChNP’s. The positively charged DOX and negatively charged siRNA encapsulated into ChNP’s. The particle size and zeta potential of the developed ChNP’s were studied by particle size analyzer and morphology was examined by TEM. Encapsulation of DOX in ChNP’s was confirmed by FTIR spectroscopy. The encapsulation efficiency and in vitro release of DOX were studied by UV-Vis spectrophotometry. The siRNA loading into ChNP’s was confirmed by gel retardation assay. Results: The developed ChNP’s showed particle size ranged from 127±6.5 to 215±8.5 nm with zeta potential ranged from 16.5±0.3 to 25.8±0.3. Transmission Electron Micrograph showed DOX and siRNA encapsulated ChNP’s are polydisperse and spherical in nature. FTIR study confirmed the binding of DOX with ChNP’s with absorption peaks at 1016 cm-1,1316 cm-1, 1412 cm-1, 1645 cm-1 and 3370 cm-1. The TPP:Ch ratio 0.1:0.5 showed the highest encapsulation efficiency 69±3.24%, with initial burst release and then sustained or slow release of DOX. Agarose gel retardation study confirmed the encapsulation of siRNA in ChNP’s by retarded migration of siRNA-ChNP’s in comparison with naked siRNA. Conclusion: The developed ChNP’s successfully encapsulated the DOX and siRNA and showed the sustain release of DOX. In conclusion, our study shown that ChNP’s is having a potential of co-loading of DOX-siRNA as an efficient drug delivery system for the treatment of various cancers such as colorectal cancer, breast cancer etc.

Effect of miR-146a/bFGF/PEG-PEI Nanoparticles on Inflammation Response and Tissue Regeneration of Human Dental Pulp Cells

Introduction. Inflammation in dental pulp cells (DPCs) initiated by Lipopolysaccharide (LPS) results in dental pulp necrosis. So far, whether there is a common system regulating inflammation response and tissue regeneration remains unknown. miR-146a is closely related to inflammation. Basic fibroblast growth factor (bFGF) is an important regulator for differentiation.Methods. To explore the effect of miR-146a/bFGF on inflammation and tissue regeneration, polyethylene glycol-polyethyleneimine (PEG-PEI) was synthesized, and physical characteristics were analyzed by dynamic light scattering and gel retardation analysis. Cell absorption, transfection efficiency, and cytotoxicity were assessed. Alginate gel was combined with miR-146a/PEG-PEI nanoparticles and bFGF. Drug release ratio was measured by ultraviolet spectrophotography. Proliferation and odontogenic differentiation of DPCs with 1 μg/mL LPS treatment were determined.Results. PEG-PEI prepared at N/P 2 showed complete gel retardation and smallest particle size and zeta potential. Transfection efficiency of PEG-PEI was higher than lipo2000. Cell viability decreased as N/P ratio increased. Drug release rate amounted to 70% at the first 12 h and then maintained slow release afterwards. Proliferation and differentiation decreased in DPCs with LPS treatment, whereas they increased in miR-146a/bFGF gel group.Conclusions. PEG-PEI is a promising vector for gene therapy. miR-146a and bFGF play critical roles in inflammation response and tissue regeneration of DPCs.

Enantioselective Regulation of Lactate Racemization by LarR in Lactobacillus plantarum

Lactobacillus plantarumis a lactic acid bacterium that produces a racemic mixture ofl- andd-lactate from sugar fermentation. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by thel-/d-lactate ratio and maximally induced in the presence ofl-lactate. We previously reported that the Lar activity depends on the expression of two divergently oriented operons: (i) thelarABCDEoperon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) thelarR(MN)QOoperon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a novel regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterizedin vivoandin vitro. We show that LarR is a positive regulator that is absolutely required for the expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in thelarR-larAintergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from thelarApromoter (PlarA). Two half-Lar boxes located between the Lar box and the −35 box of PlarApromote LarR multimerization on DNA, and point mutations within one or both half-Lar boxes inhibit PlarAinduction byl-lactate. Gel retardation and footprinting experiments indicate thatl-lactate has a positive effect on the binding and multimerization of LarR, whiled-lactate antagonizes the positive effect ofl-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.

Regulation of the Bacillus subtilis Divergent yetL and yetM Genes by a Transcriptional Repressor, YetL, in Response to Flavonoids

ABSTRACT DNA microarray analysis revealed that transcription of the Bacillus subtilis yetM gene encoding a putative flavin adenine dinucleotide-dependent monooxygenase was triggered by certain flavonoids during culture and was derepressed by disruption of the yetL gene in the opposite orientation situated immediately upstream of yetM, which encodes a putative MarR family transcriptional regulator. In vitro analyses, including DNase I footprinting and gel retardation analysis, indicated that YetL binds specifically to corresponding single sites in the divergent yetL and yetM promoter regions, with higher affinity to the yetM region; the former region overlaps the Shine-Dalgarno sequence of yetL, and the latter region contains a perfect 18-bp palindromic sequence (TAGTTAGGCGCCTAACTA). In vitro gel retardation and in vivo lacZ fusion analyses indicated that some flavonoids (kaempferol, apigenin, and luteolin) effectively inhibit YetL binding to the yetM cis sequence, but quercetin, galangin, and chrysin do not inhibit this binding, implying that the 4-hydroxyl group on the B-ring of the flavone structure is indispensable for this inhibition and that the coexistence of the 3-hydroxyl groups on the B- and C-rings does not allow antagonism of YetL.